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Cardiac myxoma (Endocardioma)
HUMAN I'ATHOLOGY--VOLUME
12, N U M B E R 10
October 1981
cidcd with ultrastrt~ctural studies o f spontaneous osteosarcomas. O u r observations were c o m p a r a b l e to those in tim literature. In light and ultrastructural study o f norreal osteoblasts and osteoblastoma, it is now widely accepted tlmt tim principal tnmor cells are osteoblasts. The dilated rough endoplasmic retictdum does not seem specific and is probably a product o f active cellular metabolism. Furthermore, every degree o f chondroid and osteoid differentiation was apparent in our osteosarcomas. The matrix showed scattered collagen fibers and fibrils without periodicity; calcium deposits and hydroxyapatite crystals were also noted with normal osteon calcification as in cartilage, embryonal bone, dentin, and callus bone. ACKNOWLEDGMENTS T h e a u t h o r s tharjk Professor A. T r i f a u d ( D e p a r t m e n t o f O r t h o p e d i c S u r g e r y , C o n c e p t i o n Hospital, Marseille) and Mrs. D. Callier for technical assistance.
REFERENCES
2. Paschall, tl. A., and Paschall, M. M.: Electron microscopic observations of 20 human osteosarcomas. Clin. Orthop., 111: 42-55, 1975. 3. Williams, A. H., Schwinn, C. P., and l'arkcr, J. W.: The ultracellular branched tubular structures in osteosarcoma. Cancer, 37:1293- 1301, 1976. 4. Reddick, R., Michelitch, H., Levine, A., and Trichc, T.: Osteogenic sarcoma. A study of the uhrastrncture. Cancer, 45:64-71, 1980. 5. ttirota, K.: EIcctnm microscopic stt,dies o n b o n e tnnmrs. II. Osteogenic sarcoma (sclerosing form). Knmamoto Med. J., 13:118-128, 1960. 6. Jenson, A. B., Spjnt, t|. J., Snfith, M. N., and Rapp, F.: lntracellular branched tubular structures in osteosarcoma. Cancer, 27:1440- 1448, 1971. 7. Ghadially, F.N., and Mehta, I'. N.: Ultrastrncturc of osteogenic sarcoma. Cancer, 25:1457-1467, 1970. 8. Kay, S.: Uhrastructure of an ostcoid type of osteogenic sarcoma. Cancer, 28:437-,t45, 1971. 9. Vuletin, J. C.: Myofibroblast in parosteal osteogenic sarcoma. Arch. Pathol. Lab. Mcd., 101:272, 1977. 10. Nilsson, A., and 8joden, I.: Uhrastructnre of 9~ induced osteosarcomas and early phases of their devr Acta Radiol. Ther. Phys. Biol. 13:107-128, 1974. 1 I. Lee, W. R., Laurie, J., and Townsend, A. L.: Fine structure of a radiation-ind uced osteogenic sarcoma. Cancer, 36:14141425, 1975.
1. tlirota, K.: Electron-microscopic studies ml bone tumors. I. Osteogenic sarcoma (sclerosing form). Knmamoto Med. J., 12:265, 1959.
Centre tlospitalier Rdgional de la Tinmne Chcmin dc l'Armde d'Afrique 13385 Marseillc, Cddex 4 France (Dr. Garbc)
CARDIAC MYXOMA (ENDOCARDIOMA) An Immunocytochemical Assessment of Histogenesis Azorides R. Morales, M.D., * Gerald Fine, M.D., ~( Albert Caslro, Ph.D., and Mehrdad Na~O'i, M.D. w Abstract Eighteen cardiac myxomas lmving a variety o f histologic patterns were stained for factor VIII related antigen utilizing the peroxidase-antiperoxidase teclmique. Regardless of their lfistologic appearance, intense reactivity for factor VIII related antigen was found in tumor cells, whereas four other intracardiac tumors did not stain. Since factor VIII related antigen is known to be elaborated only by endothelial cells add megakaryocytes, it is concluded that cardiac myxoma cells, which similarly synthesize this antigen, originate from endothelial or endocardial cells. Accepted for publication December 15, 1980. * Professor and Chairman, Department of Pathology, University of Miami-Jackson Memorial Medical Center. Chief, Pathology Services, Jackson Memorial Hospital, Miami, Florida. t Director, Division of Anatomic Pathology, Henry Ford tlospital, Detroit, Michigan. ~zProfessor of Pathology and Director, Hormone Research LaDoratory, University of Miami School of Medicine, Miami, Florida. w Associate Professor of Pathology and Director of Cytpathology, University of Miami School of Medicine. Director of Cytopathology, Jackson Memorial Medical Center, Miami, Florida.
896
C A R D I A C MYXOMA--MORALES ET AL.
Figure l. Characteristic villous surface projections of cardiac myxoma showing intense factor VIll related antigen staining of tumor cells. In this illustration lepidic cells are growing singly or forming small nests. (Peroxidase-antiperoxidase-hematoxylin stain. •
Figure 2. Interconnecting strands of tumor cells, a frequent histologic pattern in cardiac myxomas. Factor VIIIR:Ag is universally present in the cytoplasm of ttunnr cells; the acelhdar stroma is weakly reactive and nuclei only show the hematoxylin counterstaiu. (l'eroxidase-antiperoxidase-hematoxylin stain.
x400.)
Cardiac myxomas have been described for more than a century, the first possible example being that of Ornellas' lobulated left atrial tumor? Innumerable reports on this subject have followed, and although the neoplastic, as opposed to the thrombotic, nature of the tumor is almost universally accepted, its histogenesis has remained controversial. The term myxoma adequately describes the gross and microscopic aspects of the tumor, but it provides no information as to the cell of origin. Special histochemical stains, enzyme histochemistry, tissue culture, and electron microscopic studies have failed to characterize the tumor's histogenesis to everyone's satisfaction.2-s Immunocytochemical techniques have made it possible to demonstrate specific cell and tissue markers,in routinely processed pathologic material. One such marker, factor VIII related antigen (VIIIR:Ag), has been shown to be exclusively elaborated by endothelial cells and megakaryocytes.9 Since endocardial cells are a type of endothelium, we looked for the presence of VIIIR:Ag in endocardial cells and cardiac myxomas in an attempt to confirm the hypothesis that cardiac myxomas are derived from endocardial cells.
Figure 3. Tumor cells in close association with a centrally placed, thin walled blood vessel. Note intense staining of lepidic anti endothelial cell as well as plasma. (Peroxidase-antiperoxidasehematoxylin stain. •
MATERIALS AND METHODS
Formalin fixed, paraffin embedded histologic material from 18 cardiac myxomas was obtained from the files of the University of Miami-Jackson Memorial Medical Center, Miami, and Henry Ford Hospital, Detroit. Four metastatic intracardiac tumors (malignant melanoma, hepatocellular carcinoma, rhabdomyosarcoma, and anaplastic small cell carcinoma) ",aswell as two surgically removed portions of cardiac tissue, partially lined by endocardium, were also included in this study. T h e peroxidase-antiperoxidase procedure for VIIIR:Ag was carried out in one or several selected tissue blocks in a standard faslfion. Briefly~ 3 micron
Figure 4. Glandlike structure in a cardiac myxoma, showing factor VIIlR:Ag staining in the basal portion of goblet cells. (Peroxidase-antiperoxidase-hematoxylin stain. •
897
HUMAN PATHOLOGY--VOLUME 12, NUMBER 10 October 1981
9Figure 5. Groupofendocardial cells from surgicallyexcised cardiac tissue demonstratingintense reactivityfor factor VIIIR:Ag. (Pcroxidase-antiperoxidase-henmtoxylinstain. •
Antibody control in each run consisted of the use of an adjacent section treated with rabbit antiserum to VIIIR:Ag, preabsorbed by normal human plasma or substituted by nonimmune rabbit serum. Sections o f normal spleen or granulation tissue, both containing large n u m b e r s o f endothelial cells, r e p r e s e n t e d known positives for each reaction. In order to rule out possible absorption of VIIIR:Ag by myxoma cells as the mechanism for a positive reaction, in each case adjacent tissue sections were similarly stained for other plasma p r o t e i n s - - h u m a n albumin, alpha-1antitrypsin, or fibrinogen. Rabbit antiserum to factor VIII associated protein was obtained commercially (Calbiochem-Behring Corporation, La Jolla, California). This antibody was preabsorbed by either human fibrinogen or yon Willebrand plasma. Swine antirabbit immunoglobulin, rabbit peroxidase-antiperoxidase, and rabbit antisera to h u m a n a l b u m i n , f i b r i n o g e n , a n d alpha-1antitrypsin were commercially obtained (DAKO Accurate Chemical and Scientific Corporation, 28 Tec Street, Hicksville, New York). H u m a n von Willebrand plasma was generously supplied by Doctor John B. Miale, Department of Pathology, University of Miami School of Medicine, Miami, Florida.
RESULTS
Figure 6. Control section of a cardiac myxomatreated with high concentrations of antihumau albumin instead of factor VIIIR:Agantiserum.Stainingis confinedto the rnyxomatousstroma; the endotheliumofbk• vesselsand tumorcellsare distinctlynegative. (Peroxidase-antiperoxidase-hematoxylinstain, x125.)
thick sections were incubated at 58 ~ C. for one hour, deparaffinized in xylene, and then carried through decreasing grades of ethanol. After blocking the endogenous peroxidase by a solution o f h y d r o g e n peroxide and methanol, the slides were sequentially treated with nornml swine serum, rabbit antiserum to VII I R:Ag, swine antirabbit immunoglobulin, and rabbit peroxidase-antiperoxidase. The final reaction was visualized by adding diaminobenzidine in the presence of hydrogen peroxide. The optimal dilutions of antisera used in this study were as follows: rabbit antiserum to factor VIII associated protein, 1:25 to 1:200; swine antirabbit immunoglobulin, 1:10; and rabbit peroxidase-antiperoxidase, 1:500. These dilutions were calculated by checkerboard studies in which serial dilutions" o f primary antiserum were tested against several concentrations of peroxidaseantiperoxidase on mnltiple.recuts from the same tissue block.
898
Tile various histologic patterns that have been described in cardiac myxomas were all represented in the studied cases. Myxoma "lepidic" cells were found growing singly or forming small nests and interconnecting strands (Figs. 1, 2). 1~ T h e y often had a perivascular arrangement (Fig. 3). Occasionally they formed glandlike groups with a round central space; some of the cells in these formations displayed vacuolated cytoplasm mimicking goblet cells (Fig. 4). In every instance lepidic cells, singly or in groups, were lying in the midst of an abundant myxoid stroma, which stained with alcian blue and o t h e r mucopolysaccharide stains. In every one of the cardiac myxomas the lepidic cells showed i n t e n ~ reactivity for VIIIR:Ag after tile peroxidase-antiperoxidase p r o c e d u r e (Figs. 1, 4). Endocardial cells in two cases of surgically removed cardiac tissue as well as endocardial cells lining atrium adjacent to myxomas also showed an intense reactivity for VIIIR:Ag (Fig. 5). Intravascular plasma and platelets reacted positively for VIIIR:Ag as well (Fig. 3). Ill many instances focal positive staining was also observed in intervening stronm. Since this reaction was not seen in sections treated with absorbed primary antiserum, it was thought to be due to either leakage of plasma factor VIII or the antigen secreted by myxoma cells. None of the four metastatic endocardiac tumors reacted positively. Similarly the control sections treated with .antihunmn albumin, alpha-l-antitrypsin, and fibrinogen showed a positive reaction only for plasma and myxomatous stroma, whereas lepidic cells and endothelial anti endocardial cells did not stain (Fig. 6).
CARDIAC MYXOMA--MoRALES ET AL.
DISCUSSION T h e a b u n d a n t m u c o p o l y s a c c h a r i d e c o n t e n t imparts to the c a r d i a c m y x o m a an a l m o s t s t e r e o t y p e d a p p e a r a n c e with the e x c e p t i o n o f the i n f r e q n e n t case in w h i c h massive h e m o r r h a g e a n d focal calcification m a y o v e r s h a d o w the g e l a t i n o u s t e x t u r e o f the g r o w t h . W h e r e a s its m u c i n o u s s t r o m a m a y be identical to t h a t seen in a variety o f o t h e r c a r d i a c a n d e x t r a c a r d i a c mesenchymal tumors, the cellular composition of m y x o m a s a n d their a r r a n g m e n t a r e u n i q u e . Since auf f f b r s have a t t r i b u t e d the special m o r p h o l o g i c c h a r acteristics o f the c a r d i a c m y x o m a to the i m p a c t o f h e m o d y n a m i c forces in i n t r a c a v i t a r y masses; o t h e r s c o n s i d e r t h e m to be d u e to o r g a n i z e d t h r o m b i , n I n a d d i t i o n , a variety o f o p i n i o n s have b e e n exp r e s s e d as to the f u n d a m e n t a l n a t u r e o f the cells t h a t c o m p o s e t h e m y x o i n a . Stein et al., G S i l v e r b e r g a n d K a y / a n d m o r e recently R o b e r t s a n d F e r r a n s s in t h e i r e l e c t r o n m i c r o s c o p i c studies d e s c r i b e d a variety o f d i f f e r e n t neoplastic cells ( v a s o f o r m a t i v e a n d s m o o t h muscle) in m y x o m a s a n d c o n c l u d e d t h a t these t u n m r s arise f r o m m u l t i p o t e n t i a l s u b e n d o c a r d i a i r e s e r v e cells. It shottld be p o i n t e d out, h o w e v e r , t h a t the heterogeneous cellular morphology f o t t n d b y ult r a s t r u c t u r a l studies o f c a r d i a c m y x o m a s is sltared by a number of benign and malignant neoplasms. B e c a u s e o f the alkaline p h o s p l m t a s e c o n t e n t o f the t u n a o r cells a n d o t h e r histologic findings, a Fine, Morales, a n d H o r n 3 f a v o r e d t h e view t h a t c a r d i a c m y x o m a s are d e r i v e d f r o m e n d o t h e l i u m o r e n d o c a r d i u m . T h e y d e s c r i b e d the histologic variability, n o t e d p r e v i o u s l y by o t h e r s , e m p h a s i z i n g the epithelial a n d glandlike a r r a n g e m e n t s , a n d e x p r e s s e d t h e o p i n i o n t h a t s u c h g r o u p i n g s arise f r o m the s a m e e n d o t h e l i a l o r i g i n s as the m y x o m a cells themselves. As with o t h e r t u m o i ' s d e r i v e d f r o m e n d o t h e l i t m L t2 the p r e s e n c e o f f a c t o r V I I I related a n t i g e n in the c a r d i a c m y x o m a can be c o n s i d e r e d to be conclnsive e v i d e n c e o f an e n d o t h e l i a l o r e n d o c a r d i a l n a t u r e o f t u m o r cells. It is n o t possible, h o w e v e r , to establish w h e t h e r the lepidic cells d e r i v e f r o m primitive cells o r f r o m p r o l i f e r a t i o n o f e n d o t h e l i a l o r e n d o c a r d i a l cells. T h e possibility o f a b s o r p t i o n o f V I I I R : A g by m y x o m a cells was r u l e d o u t by s i m u h a n e o u s n e g a t i v e s t a i n i n g f o r o t h e r p l a s m a proteins, a n d t h e r e f o r e it b e c o m e s e v i d e n t
t h a t lepidic cells are actively involved in the synthesis o f V I I I R : A g , akin to n o r m a l e n d o t h e l i a l cells. I n light o f o u r f i n d i n g s , it m i g h t be logical to a s s u m e t h a t t h e descriptive t e r m " c a r d i a c m y x o m a " can be r e p l a c e d by a histogenetically a p p r o p r i a t e designation, s u c h as c a r d i a c e n d o t h e l i o m a or, as we prefer, "endocardioma."
ACKNOWLEDGMENT The technical assistance o f Leslie Freedman is gratefully acknowledged.
REFERENCES 1. Ornellas: Grail tumor fibroso en la ,mricula izquierda del corazon. Gaceta Medica de Lima, 15:271, 1858. 2. Fine, G.: Neoplasms of tile pericardiunl aud heart. In Gould, S. E. (Editor): Pathology of the Heart and Blood Vessels. Ed. 3. Springfickl, Illinois, Charles C Thomas, l'ublisher, pp. 851-881. 3. Fine, G., Morales, A. R., and Horn, R. C.: Cardiac myxoma. A morphologic and histogenetic appraisal. Cancer, 22:1156, 1968. 4. Morales, A. R., Fine, G., Riddle, J. M., and Horn, R. C.: Enzyme histochcmistry and clectrcm microscopic stud)' of cardiac myxoma. Amer. J. Clin. l'ath., 44:572, 1965. 5. Glasser, S. 1'., Bcdyncck, J. L., l iall, R.J., Hopeman, A. R., Treasure, R. L., McAllistcr, tt. A., Estcrly, J. A., Man|on, W. C., and Stanfi~rd, H.S.: Left atrial myxoma. Amcr. J. Med., 50:113, 1971. 6. Stein, A. A., Mauro, J., Thiboden, L., and Alley, R.: The histogencsis of cardiac myxomas: relation to other proliferative diseases of subcndothcliaI vaso-fi,rm reserve cells. In Sommcrs, S.C. (editor): l'athology Annual. New York, Appleton-Century-Crofts, 1969, Vol. 4, pp. 293- 312. 7. Silvcrberg, S. G., and Kay, S.: Uhrastructurc of a cardiac myxoma. Amer..l- Clin. l'ath., 54:650, 1970. 8. Ferrans, V. S., and Roberts, W. C.: Strt,ctural features of cardiac nlyxolna. Histology, histochemistry, and electron microscopy. Human Path., 4:11 i, 1973. 9. Jaffe, E. A.: Endothelial cells and the biology of factor VIII. N. Engl. J. Med., 296:377, 1977. 10. Orr, J. w.: Endothelioma (psetldolnyxonla) of tile heart. J. Path. Bact., 54:125, 1942. 11. Salyer, W. R., Page, D. L., and Hutchins, G. M.: The developmerit of cardiac myxomas and papillary end,,cardial lesions from mural thr,,mbus. Amer. Heart J., 89:4, 1975. 12. Nadji, M., Gonzalez, M.S., Castro, A., and Morales, A. R.: Factor VIII related antigen: an endothelial cell marker. Lab. Invest., 42:139, 1980. Department of l'athology University of Miami School of Medicine P. O. Box 016960 Miami, Florida 33101 (Dr. Morales)
899
12, N U M B E R 10
October 1981
cidcd with ultrastrt~ctural studies o f spontaneous osteosarcomas. O u r observations were c o m p a r a b l e to those in tim literature. In light and ultrastructural study o f norreal osteoblasts and osteoblastoma, it is now widely accepted tlmt tim principal tnmor cells are osteoblasts. The dilated rough endoplasmic retictdum does not seem specific and is probably a product o f active cellular metabolism. Furthermore, every degree o f chondroid and osteoid differentiation was apparent in our osteosarcomas. The matrix showed scattered collagen fibers and fibrils without periodicity; calcium deposits and hydroxyapatite crystals were also noted with normal osteon calcification as in cartilage, embryonal bone, dentin, and callus bone. ACKNOWLEDGMENTS T h e a u t h o r s tharjk Professor A. T r i f a u d ( D e p a r t m e n t o f O r t h o p e d i c S u r g e r y , C o n c e p t i o n Hospital, Marseille) and Mrs. D. Callier for technical assistance.
REFERENCES
2. Paschall, tl. A., and Paschall, M. M.: Electron microscopic observations of 20 human osteosarcomas. Clin. Orthop., 111: 42-55, 1975. 3. Williams, A. H., Schwinn, C. P., and l'arkcr, J. W.: The ultracellular branched tubular structures in osteosarcoma. Cancer, 37:1293- 1301, 1976. 4. Reddick, R., Michelitch, H., Levine, A., and Trichc, T.: Osteogenic sarcoma. A study of the uhrastrncture. Cancer, 45:64-71, 1980. 5. ttirota, K.: EIcctnm microscopic stt,dies o n b o n e tnnmrs. II. Osteogenic sarcoma (sclerosing form). Knmamoto Med. J., 13:118-128, 1960. 6. Jenson, A. B., Spjnt, t|. J., Snfith, M. N., and Rapp, F.: lntracellular branched tubular structures in osteosarcoma. Cancer, 27:1440- 1448, 1971. 7. Ghadially, F.N., and Mehta, I'. N.: Ultrastrncturc of osteogenic sarcoma. Cancer, 25:1457-1467, 1970. 8. Kay, S.: Uhrastructure of an ostcoid type of osteogenic sarcoma. Cancer, 28:437-,t45, 1971. 9. Vuletin, J. C.: Myofibroblast in parosteal osteogenic sarcoma. Arch. Pathol. Lab. Mcd., 101:272, 1977. 10. Nilsson, A., and 8joden, I.: Uhrastructnre of 9~ induced osteosarcomas and early phases of their devr Acta Radiol. Ther. Phys. Biol. 13:107-128, 1974. 1 I. Lee, W. R., Laurie, J., and Townsend, A. L.: Fine structure of a radiation-ind uced osteogenic sarcoma. Cancer, 36:14141425, 1975.
1. tlirota, K.: Electron-microscopic studies ml bone tumors. I. Osteogenic sarcoma (sclerosing form). Knmamoto Med. J., 12:265, 1959.
Centre tlospitalier Rdgional de la Tinmne Chcmin dc l'Armde d'Afrique 13385 Marseillc, Cddex 4 France (Dr. Garbc)
CARDIAC MYXOMA (ENDOCARDIOMA) An Immunocytochemical Assessment of Histogenesis Azorides R. Morales, M.D., * Gerald Fine, M.D., ~( Albert Caslro, Ph.D., and Mehrdad Na~O'i, M.D. w Abstract Eighteen cardiac myxomas lmving a variety o f histologic patterns were stained for factor VIII related antigen utilizing the peroxidase-antiperoxidase teclmique. Regardless of their lfistologic appearance, intense reactivity for factor VIII related antigen was found in tumor cells, whereas four other intracardiac tumors did not stain. Since factor VIII related antigen is known to be elaborated only by endothelial cells add megakaryocytes, it is concluded that cardiac myxoma cells, which similarly synthesize this antigen, originate from endothelial or endocardial cells. Accepted for publication December 15, 1980. * Professor and Chairman, Department of Pathology, University of Miami-Jackson Memorial Medical Center. Chief, Pathology Services, Jackson Memorial Hospital, Miami, Florida. t Director, Division of Anatomic Pathology, Henry Ford tlospital, Detroit, Michigan. ~zProfessor of Pathology and Director, Hormone Research LaDoratory, University of Miami School of Medicine, Miami, Florida. w Associate Professor of Pathology and Director of Cytpathology, University of Miami School of Medicine. Director of Cytopathology, Jackson Memorial Medical Center, Miami, Florida.
896
C A R D I A C MYXOMA--MORALES ET AL.
Figure l. Characteristic villous surface projections of cardiac myxoma showing intense factor VIll related antigen staining of tumor cells. In this illustration lepidic cells are growing singly or forming small nests. (Peroxidase-antiperoxidase-hematoxylin stain. •
Figure 2. Interconnecting strands of tumor cells, a frequent histologic pattern in cardiac myxomas. Factor VIIIR:Ag is universally present in the cytoplasm of ttunnr cells; the acelhdar stroma is weakly reactive and nuclei only show the hematoxylin counterstaiu. (l'eroxidase-antiperoxidase-hematoxylin stain.
x400.)
Cardiac myxomas have been described for more than a century, the first possible example being that of Ornellas' lobulated left atrial tumor? Innumerable reports on this subject have followed, and although the neoplastic, as opposed to the thrombotic, nature of the tumor is almost universally accepted, its histogenesis has remained controversial. The term myxoma adequately describes the gross and microscopic aspects of the tumor, but it provides no information as to the cell of origin. Special histochemical stains, enzyme histochemistry, tissue culture, and electron microscopic studies have failed to characterize the tumor's histogenesis to everyone's satisfaction.2-s Immunocytochemical techniques have made it possible to demonstrate specific cell and tissue markers,in routinely processed pathologic material. One such marker, factor VIII related antigen (VIIIR:Ag), has been shown to be exclusively elaborated by endothelial cells and megakaryocytes.9 Since endocardial cells are a type of endothelium, we looked for the presence of VIIIR:Ag in endocardial cells and cardiac myxomas in an attempt to confirm the hypothesis that cardiac myxomas are derived from endocardial cells.
Figure 3. Tumor cells in close association with a centrally placed, thin walled blood vessel. Note intense staining of lepidic anti endothelial cell as well as plasma. (Peroxidase-antiperoxidasehematoxylin stain. •
MATERIALS AND METHODS
Formalin fixed, paraffin embedded histologic material from 18 cardiac myxomas was obtained from the files of the University of Miami-Jackson Memorial Medical Center, Miami, and Henry Ford Hospital, Detroit. Four metastatic intracardiac tumors (malignant melanoma, hepatocellular carcinoma, rhabdomyosarcoma, and anaplastic small cell carcinoma) ",aswell as two surgically removed portions of cardiac tissue, partially lined by endocardium, were also included in this study. T h e peroxidase-antiperoxidase procedure for VIIIR:Ag was carried out in one or several selected tissue blocks in a standard faslfion. Briefly~ 3 micron
Figure 4. Glandlike structure in a cardiac myxoma, showing factor VIIlR:Ag staining in the basal portion of goblet cells. (Peroxidase-antiperoxidase-hematoxylin stain. •
897
HUMAN PATHOLOGY--VOLUME 12, NUMBER 10 October 1981
9Figure 5. Groupofendocardial cells from surgicallyexcised cardiac tissue demonstratingintense reactivityfor factor VIIIR:Ag. (Pcroxidase-antiperoxidase-henmtoxylinstain. •
Antibody control in each run consisted of the use of an adjacent section treated with rabbit antiserum to VIIIR:Ag, preabsorbed by normal human plasma or substituted by nonimmune rabbit serum. Sections o f normal spleen or granulation tissue, both containing large n u m b e r s o f endothelial cells, r e p r e s e n t e d known positives for each reaction. In order to rule out possible absorption of VIIIR:Ag by myxoma cells as the mechanism for a positive reaction, in each case adjacent tissue sections were similarly stained for other plasma p r o t e i n s - - h u m a n albumin, alpha-1antitrypsin, or fibrinogen. Rabbit antiserum to factor VIII associated protein was obtained commercially (Calbiochem-Behring Corporation, La Jolla, California). This antibody was preabsorbed by either human fibrinogen or yon Willebrand plasma. Swine antirabbit immunoglobulin, rabbit peroxidase-antiperoxidase, and rabbit antisera to h u m a n a l b u m i n , f i b r i n o g e n , a n d alpha-1antitrypsin were commercially obtained (DAKO Accurate Chemical and Scientific Corporation, 28 Tec Street, Hicksville, New York). H u m a n von Willebrand plasma was generously supplied by Doctor John B. Miale, Department of Pathology, University of Miami School of Medicine, Miami, Florida.
RESULTS
Figure 6. Control section of a cardiac myxomatreated with high concentrations of antihumau albumin instead of factor VIIIR:Agantiserum.Stainingis confinedto the rnyxomatousstroma; the endotheliumofbk• vesselsand tumorcellsare distinctlynegative. (Peroxidase-antiperoxidase-hematoxylinstain, x125.)
thick sections were incubated at 58 ~ C. for one hour, deparaffinized in xylene, and then carried through decreasing grades of ethanol. After blocking the endogenous peroxidase by a solution o f h y d r o g e n peroxide and methanol, the slides were sequentially treated with nornml swine serum, rabbit antiserum to VII I R:Ag, swine antirabbit immunoglobulin, and rabbit peroxidase-antiperoxidase. The final reaction was visualized by adding diaminobenzidine in the presence of hydrogen peroxide. The optimal dilutions of antisera used in this study were as follows: rabbit antiserum to factor VIII associated protein, 1:25 to 1:200; swine antirabbit immunoglobulin, 1:10; and rabbit peroxidase-antiperoxidase, 1:500. These dilutions were calculated by checkerboard studies in which serial dilutions" o f primary antiserum were tested against several concentrations of peroxidaseantiperoxidase on mnltiple.recuts from the same tissue block.
898
Tile various histologic patterns that have been described in cardiac myxomas were all represented in the studied cases. Myxoma "lepidic" cells were found growing singly or forming small nests and interconnecting strands (Figs. 1, 2). 1~ T h e y often had a perivascular arrangement (Fig. 3). Occasionally they formed glandlike groups with a round central space; some of the cells in these formations displayed vacuolated cytoplasm mimicking goblet cells (Fig. 4). In every instance lepidic cells, singly or in groups, were lying in the midst of an abundant myxoid stroma, which stained with alcian blue and o t h e r mucopolysaccharide stains. In every one of the cardiac myxomas the lepidic cells showed i n t e n ~ reactivity for VIIIR:Ag after tile peroxidase-antiperoxidase p r o c e d u r e (Figs. 1, 4). Endocardial cells in two cases of surgically removed cardiac tissue as well as endocardial cells lining atrium adjacent to myxomas also showed an intense reactivity for VIIIR:Ag (Fig. 5). Intravascular plasma and platelets reacted positively for VIIIR:Ag as well (Fig. 3). Ill many instances focal positive staining was also observed in intervening stronm. Since this reaction was not seen in sections treated with absorbed primary antiserum, it was thought to be due to either leakage of plasma factor VIII or the antigen secreted by myxoma cells. None of the four metastatic endocardiac tumors reacted positively. Similarly the control sections treated with .antihunmn albumin, alpha-l-antitrypsin, and fibrinogen showed a positive reaction only for plasma and myxomatous stroma, whereas lepidic cells and endothelial anti endocardial cells did not stain (Fig. 6).
CARDIAC MYXOMA--MoRALES ET AL.
DISCUSSION T h e a b u n d a n t m u c o p o l y s a c c h a r i d e c o n t e n t imparts to the c a r d i a c m y x o m a an a l m o s t s t e r e o t y p e d a p p e a r a n c e with the e x c e p t i o n o f the i n f r e q n e n t case in w h i c h massive h e m o r r h a g e a n d focal calcification m a y o v e r s h a d o w the g e l a t i n o u s t e x t u r e o f the g r o w t h . W h e r e a s its m u c i n o u s s t r o m a m a y be identical to t h a t seen in a variety o f o t h e r c a r d i a c a n d e x t r a c a r d i a c mesenchymal tumors, the cellular composition of m y x o m a s a n d their a r r a n g m e n t a r e u n i q u e . Since auf f f b r s have a t t r i b u t e d the special m o r p h o l o g i c c h a r acteristics o f the c a r d i a c m y x o m a to the i m p a c t o f h e m o d y n a m i c forces in i n t r a c a v i t a r y masses; o t h e r s c o n s i d e r t h e m to be d u e to o r g a n i z e d t h r o m b i , n I n a d d i t i o n , a variety o f o p i n i o n s have b e e n exp r e s s e d as to the f u n d a m e n t a l n a t u r e o f the cells t h a t c o m p o s e t h e m y x o i n a . Stein et al., G S i l v e r b e r g a n d K a y / a n d m o r e recently R o b e r t s a n d F e r r a n s s in t h e i r e l e c t r o n m i c r o s c o p i c studies d e s c r i b e d a variety o f d i f f e r e n t neoplastic cells ( v a s o f o r m a t i v e a n d s m o o t h muscle) in m y x o m a s a n d c o n c l u d e d t h a t these t u n m r s arise f r o m m u l t i p o t e n t i a l s u b e n d o c a r d i a i r e s e r v e cells. It shottld be p o i n t e d out, h o w e v e r , t h a t the heterogeneous cellular morphology f o t t n d b y ult r a s t r u c t u r a l studies o f c a r d i a c m y x o m a s is sltared by a number of benign and malignant neoplasms. B e c a u s e o f the alkaline p h o s p l m t a s e c o n t e n t o f the t u n a o r cells a n d o t h e r histologic findings, a Fine, Morales, a n d H o r n 3 f a v o r e d t h e view t h a t c a r d i a c m y x o m a s are d e r i v e d f r o m e n d o t h e l i u m o r e n d o c a r d i u m . T h e y d e s c r i b e d the histologic variability, n o t e d p r e v i o u s l y by o t h e r s , e m p h a s i z i n g the epithelial a n d glandlike a r r a n g e m e n t s , a n d e x p r e s s e d t h e o p i n i o n t h a t s u c h g r o u p i n g s arise f r o m the s a m e e n d o t h e l i a l o r i g i n s as the m y x o m a cells themselves. As with o t h e r t u m o i ' s d e r i v e d f r o m e n d o t h e l i t m L t2 the p r e s e n c e o f f a c t o r V I I I related a n t i g e n in the c a r d i a c m y x o m a can be c o n s i d e r e d to be conclnsive e v i d e n c e o f an e n d o t h e l i a l o r e n d o c a r d i a l n a t u r e o f t u m o r cells. It is n o t possible, h o w e v e r , to establish w h e t h e r the lepidic cells d e r i v e f r o m primitive cells o r f r o m p r o l i f e r a t i o n o f e n d o t h e l i a l o r e n d o c a r d i a l cells. T h e possibility o f a b s o r p t i o n o f V I I I R : A g by m y x o m a cells was r u l e d o u t by s i m u h a n e o u s n e g a t i v e s t a i n i n g f o r o t h e r p l a s m a proteins, a n d t h e r e f o r e it b e c o m e s e v i d e n t
t h a t lepidic cells are actively involved in the synthesis o f V I I I R : A g , akin to n o r m a l e n d o t h e l i a l cells. I n light o f o u r f i n d i n g s , it m i g h t be logical to a s s u m e t h a t t h e descriptive t e r m " c a r d i a c m y x o m a " can be r e p l a c e d by a histogenetically a p p r o p r i a t e designation, s u c h as c a r d i a c e n d o t h e l i o m a or, as we prefer, "endocardioma."
ACKNOWLEDGMENT The technical assistance o f Leslie Freedman is gratefully acknowledged.
REFERENCES 1. Ornellas: Grail tumor fibroso en la ,mricula izquierda del corazon. Gaceta Medica de Lima, 15:271, 1858. 2. Fine, G.: Neoplasms of tile pericardiunl aud heart. In Gould, S. E. (Editor): Pathology of the Heart and Blood Vessels. Ed. 3. Springfickl, Illinois, Charles C Thomas, l'ublisher, pp. 851-881. 3. Fine, G., Morales, A. R., and Horn, R. C.: Cardiac myxoma. A morphologic and histogenetic appraisal. Cancer, 22:1156, 1968. 4. Morales, A. R., Fine, G., Riddle, J. M., and Horn, R. C.: Enzyme histochcmistry and clectrcm microscopic stud)' of cardiac myxoma. Amer. J. Clin. l'ath., 44:572, 1965. 5. Glasser, S. 1'., Bcdyncck, J. L., l iall, R.J., Hopeman, A. R., Treasure, R. L., McAllistcr, tt. A., Estcrly, J. A., Man|on, W. C., and Stanfi~rd, H.S.: Left atrial myxoma. Amcr. J. Med., 50:113, 1971. 6. Stein, A. A., Mauro, J., Thiboden, L., and Alley, R.: The histogencsis of cardiac myxomas: relation to other proliferative diseases of subcndothcliaI vaso-fi,rm reserve cells. In Sommcrs, S.C. (editor): l'athology Annual. New York, Appleton-Century-Crofts, 1969, Vol. 4, pp. 293- 312. 7. Silvcrberg, S. G., and Kay, S.: Uhrastructurc of a cardiac myxoma. Amer..l- Clin. l'ath., 54:650, 1970. 8. Ferrans, V. S., and Roberts, W. C.: Strt,ctural features of cardiac nlyxolna. Histology, histochemistry, and electron microscopy. Human Path., 4:11 i, 1973. 9. Jaffe, E. A.: Endothelial cells and the biology of factor VIII. N. Engl. J. Med., 296:377, 1977. 10. Orr, J. w.: Endothelioma (psetldolnyxonla) of tile heart. J. Path. Bact., 54:125, 1942. 11. Salyer, W. R., Page, D. L., and Hutchins, G. M.: The developmerit of cardiac myxomas and papillary end,,cardial lesions from mural thr,,mbus. Amer. Heart J., 89:4, 1975. 12. Nadji, M., Gonzalez, M.S., Castro, A., and Morales, A. R.: Factor VIII related antigen: an endothelial cell marker. Lab. Invest., 42:139, 1980. Department of l'athology University of Miami School of Medicine P. O. Box 016960 Miami, Florida 33101 (Dr. Morales)
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